ISSN:
1573-5168
Keywords:
salmonid
;
sperm
;
membrane
;
nitrogen cavitation
;
electron microscopy
;
protein
;
enzyme
;
lipid
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract The plasma membrane from spermatozoa of rainbow trout was isolated by four techniques: sonication, hypotonic shock, mechanical homogenization after freeze-thawing, and nitrogen cavitation, in combination with continuous sucrose gradient centrifugation. Nitrogen cavitation (900 PSI, 20 min equilibration at 4°C) was the most effective technique. Following nitrogen cavitation, four bands were recovered in the sucrose gradient at densities ≈ 1.03, 1.05, 1.09 and 1.15 g/ml. Electron microscopy revealed membrane vesicles of various sizes in bands 1 to 3, while enzyme analysis revealed a 3.9 to 5.5-fold enrichment in 5'-nucleotidase and little contamination by lactate dehydrogenase (cytosol) and succinic dehydrogenase (mitochondria). Lipid analysis of bands 1 and 2 indicated a 6 to 7-fold enrichment in cholesterol and a cholesterol: phospholipid ratio of 0.59–0.70. Seven classes of phospholipids were present in bands 1–3 with no significant differences observed among bands. These data indicate that the vesicles (in bands 1 and 2) obtained after nitrogen cavitation are primarily plasma membranes. Membranes in band 3 appear to be slightly contaminated with nuclear membranes. Most of the plasma membrane proteins were acidic to neutral. The 2 main membrane proteins were 42 and 30 Kilodaltons.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF02265153
